Engineering bacteria of inactivated acetolactate synthetase, and applications thereof in producing 1,3-propanediol

A technology of acetolactate and synthase, applied in bacteria, microorganisms, microorganism-based methods, etc., can solve problems such as unknown impact, and achieve the effects of reducing post-extraction pressure, improving conversion rate, and reducing production costs.

Inactive Publication Date: 2013-09-18
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

But the effect of inactivation of acetolactate synthase, a key enzyme in the metabolic pathway of 2,3-butanediol, on the fermentative production of 1,3-propanediol by Klebsiella is unknown

Method used

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  • Engineering bacteria of inactivated acetolactate synthetase, and applications thereof in producing 1,3-propanediol
  • Engineering bacteria of inactivated acetolactate synthetase, and applications thereof in producing 1,3-propanediol
  • Engineering bacteria of inactivated acetolactate synthetase, and applications thereof in producing 1,3-propanediol

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Embodiment 1

[0025] 1. Construction of engineered bacteria with inactivated acetolactate synthase gene inactivated in 2,3-butanediol metabolic pathway

[0026] (l), cloning of acetolactate synthase gene ALS partial sequence

[0027]Primers were designed according to the complete DNA sequence of the acetolactate synthase gene ALS (GenBank number: YP_006633876.1), and its partial homologous sequence was amplified by PCR. . Using the genomic DNA of wild-type Klebsiella pneumoniae (CCTCC M2011075) as a template, under the guidance of primers ALS-F and ALS-R, the partial sequence of the acetolactate synthase gene ALS was amplified by PCR. The PCR amplification conditions were as follows: First 95°C for 3min; then 94°C for 1min, 55°C for 1min, 72°C for 2min, a total of 33 cycles; finally 72°C for 10min. After the reaction, the PCR amplification product was subjected to 1.0% agarose gel electrophoresis, and the target fragment of about 1500bp was recovered and purified ( figure 1 ), clone it i...

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Abstract

The invention discloses an engineering bacteria of inactivated acetolactate synthetase, and a construction method and applications thereof in producing 1,3-propanediol. The acetolactate synthetase in wild type strain for producing 1,3-propanediol is silenced by utilizing homologous recombination and gene insertion inactivation methods, thus obtaining 2,3-butanediol metabolic pathyway-blocked engineering bacteria. By using the engineering bacteria to ferment and produce 1,3-propanediol, the production of side product 2,3-butanediol can be greatly reduced, the metabolism shunting effect of the side product 2,3-butanediol can be greatly decreased, and the conversion rate for producing 1,3-propanediol can be improved; in addition, the side product 2,3-butanediol is reduced to lower the post-extraction pressure, so that the production cost can be lowered. Proved by experiments, the concentration of 1,3-propanediol can achieve 72g / L by fermenting the engineering bacteria for 36 hours through adopting a conventional method. The engineering bacteria and the construction method and applications thereof can play an important role in the industrial production of 1,3-propanediol through adopting a microbial fermentation method, and have wide application prospects.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to an engineering bacterium for inactivating acetolactate synthetase, its construction method and its application in the production of 1,3-propanediol. Background technique: [0002] 1,3-Propanediol (PDO) is an important chemical raw material. PDO can be used as an organic solvent in high pressure lubricants, dyes, inks, antifreeze and other industries; it can also be used to synthesize heterocycles, pharmaceutical intermediates, polyesters and polyurethanes; the most important use is as a monomer to synthesize polymers. Propylene Terephthalate (PTT). PTT is a new fiber-forming polyester polymer material that has achieved industrial scale after polyethylene terephthalate (PET) in the 1950s and polybutylene terephthalate (PBT) in the 1970s. A very promising new polyester material. In 1998, PTT was rated as one of the six new petrochemical products by the United States. C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/03C12N1/21C12P7/18C12R1/22C12R1/19
Inventor 周胜秦启伟李莉莉黄友华高任
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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